DESCRIPTION: The MHC class I related receptor FcRn plays an important role in the delivery of maternal gamma globulins (IgGs) to offspring during pregnancy and in maintaining serum IgG levels. It transports IgGs within and across cells. Changing the affinity of the IgG-FcRn interaction by engineering the interaction site has a significant influence on serum persistence and maternal-fetal transport of IgGs. The effective use of IgGs in passive-immunization is of major importance in situations where immediate protection against a pathogen or toxin is needed (e.g., RSV, bioterrorism agents). Understanding intracellular trafficking of IgGs and its dependence on FcRn is, aside from the biological interest, also crucial to make further progress in the development and administration of engineered antibodies with desired behavior as regards to serum persistence, maternal fetal transfer, dosage requirements, etc. One of the goals of the parent proposal is to improve on the as yet very limited understanding of intracellular trafficking of IgGs, and its dependence on FcRn using fluorescence microscopy techniques. Fluorescence microscopy of live cell samples is both a rapidly evolving technology and has been shown to be a tool which can provide significant insights in cell biological investigations. This proposal seeks to significantly upgrade the fluorescence microscopy imaging capability of the laboratory of the co-PI who is the PI on the parent proposal. The goal is to make available very advanced capabilities to the study the IgG trafficking problem. In particular, funds are sought for the purchase of additional imaging equipment, the integration of the new equipment with the existing imaging facility, the development of custom acquisition and analysis software, and its testing on key experiments arising from the investigations on the parent proposal. The first specific aim is to implement instrumentation and software for high speed dual color fluorescence microscopy. The second specific aim is to implement instrumentation and software for total internal reflection microscopy imaging and analysis. The objective of the second specific aim is to have the ability to perform experiments with single molecule detection sensitivity, such as the study of IgG exocytosis. The approach in this proposal is based on recent advances in imaging technology and its applications to cellular biology problems, in particular imaging with single molecule sensitivity.